Undergraduate Student Stipend and Research Cost Awards for Faculty-Student Collaboration

Lauren Styczynski and Mark L. Baccei, PhD

Statement of the problem:

Currently, there is a high survival rate of patients that are administered vincristine, however it remains
unknown how these drugs may exert long term effects on developing neuronal networks, like those that
are underlying pain perception. Early life tissue damage is known to exacerbate pain sensitivity following
subsequent injury, but it is still unclear if early life chemotherapy can evoke a similar “priming”. The
primary goal of this project is to understand the degree to which vincristine (a common
chemotherapeutic agent), administered in early life, primes the developing pain pathway when
subsequent injury occurs in adulthood.

Background:
Vincristine (VNC) is commonly used to treat pediatric cancers, including acute lymphoblastic leukemia
(ALL), the most common cancer for pediatric patients. While there has been an increase in the survival
rate of ALL, patients that receive VNC commonly experience an increase of pain sensitivity in their hands
and feet, which leads to a lower quality of life. Our recent work has shown that early life exposure of
high doses (60 ug/kg) of VNC in the developing rat can lead to a selective increase in mechanical pain
sensitivity that emerges during adolescence (Schappacher), thereby providing the first preclinical model
of pediatric VNC-evoked neuropathic pain.
Pediatric pain is a significant clinical problem that impacts up to 20% of children and costs an
expenditure of $19.5 billion annually. Studies have shown that neonatal pain pathways are organized
differently compared to adulthood. Ongoing developmental changes in nociceptive circuits may make
them susceptible to persistent reorganization following aberrant sensory input during early life. For
example, it is known that neonatal tissue damage, which commonly occurs in the NICU, can “prime”
pain networks in the rodent CNS and alter pain perception throughout life.
Nonetheless, whether VNC administered in early life can similarly “prime” developing pain pathways,
and thus exacerbate pain hypersensitivity following subsequent injury during adulthood, remains
unknown. This represents an important gap in our knowledge base that must be addressed before one
can fully understand how adult pain sensitivity may be influenced by a diversity of noxious sensory
experiences during early life.
In conclusion, the available evidence clearly demonstrates the need to identify the long-term effect of
VNC on sensory processing within pain circuits. Such information is also critically important as it could
provide insight into the lasting effects of VNC on the developing CNS.
Therefore, the goal of this study is to identify the degree to which neonatal VNC persistently modulates
mechanical and thermal pain hypersensitivity following tissue injury during adulthood.

Specific Aims, Research Questions or Hypotheses:

Our central hypothesis is that VNC primes developing pain circuits, thereby resulting in a higher pain
sensitivity following injury during adulthood. The central hypothesis will be tested by pursuing two
specific aims:
 1. Identify the extent to which vincristine will affect mechanical pain hypersensitivity following
adult injury.
Our working hypothesis is that exposure to high doses of VNC during early life will exacerbate
the hypersensitivity to noxious mechanical stimuli following hindpaw incision in adulthood.
1. Identify the extent to which vincristine will affect thermal pain hypersensitivity following adult
injury.
Our working hypothesis is that exposure to high doses of VNC during early life will enhance the
severity of thermal hypersensitivity following adult hindpaw incision.

Research Strategy or Plan:

DESIGN
In this study, male and female Sprague Dawley rats will be used, and randomly assigned to one of two
groups: vehicle or vincristine. Animals will be weighed prior to each injection, and then once per week
after treatment ended. For consistency with preliminary dose response studies, animals will receive 5
total doses of 60 ug/kg in saline, on days (postnatal day) P11, 13, 17, 19 and 21. Following these
injections, there will be a hindpaw incision injury in adulthood, at P64.
Three behavioral assays will be conducted to evaluate mechanical sensitivity: von Frey, paintbrush, and
pin prick tests. The von Frey test evaluates for mechanical reflex withdrawal thresholds, and will be
tested according to the SUDO method. The paintbrush test and pinprick tests will be used as measures
of dynamic mechanical allodynia and static mechanical hyperalgesia, respectively.
To test for thermal sensitivity, the Hargreaves test and the cold pantar assay will be used measure
sensitivity to noxious heat and cold, respectively, by an investigator blinded to the treatment group.
Each animal will not be subject to every behavioral assay in order to reduce stress, and will not undergo
more than two behavioral assays per day to reduce stress inflicted upon each animal.

STATISTICAL ANALYSIS
Behavioral data is to be analyzed using two-way analysis of variance (ANOVA) with Holm-Sidak post hoc
tests with drug and injury as factors.

EXPECTED OUTCOMES
Given the knowledge that VNC administered during early life evokes a delayed mechanical
hypersensitivity, it is anticipated that incision will evoke mechanical hypersensitivity of a longer duration
in those animals that received the VNC treatment as opposed to animals that received the saline control
injections. Additionally, in regards to thermal hypersensitivity, given the hypothesis that VNC selectively
increases mechanical pain sensitivity, it is anticipated that neonatal VNC will fail to alter the sensitivity
to thermal stimuli following adult surgical injury.

POTENTIAL PROBLEMS AND ALTERNATE APPROACHES

In the unlikely event that we do not see prolonged mechanical hypersensitivity in animals receiving the
VNC treatment, we will first ensure that our dosing method is correct. Once ensured that the dosing
method is indeed correct, we would then test for the effects of the VNC regimen and subsequent injury
on motor function of the animals. This would be achieved by performing two motor tests: the rotarod
test and the foot fault test. The rotarod tests for gross motor function, and the foot fault tests for fine
motor function.

Faculty Collaborator’s Role:

Dr. Baccei will conduct bi-weekly meetings with Lauren to ensure that she is on track to complete her
project on time, and on track to meet her goals. Dr. Baccei will also ensure that Lauren is trained in the
above behavioral assays of pain sensitivity, provide feedback on her choice of statistical analysis, offer
guidance in the presentation of the experimental results, and help to interpret the data in the wider
context of the pain research field.

Timeline:
The researcher should discuss when the various stages of the project will be undertaken and completed.
Include both the student’s and the faculty collaborator’s activities.
May-June: collect data for mechanical assays, perform statistical analysis
July-July: collect data for thermal assays, perform statistical analysis, and then begin writing the final
paper.

References:
List all references cited in the proposal. This section is not part of the four-page maximum for the
proposal narrative.

Brain 2012 paper - Beggs & Fitzgerald

EJN 2014 - Schwaller & Fitzgerald
PNAS 1999 - Fitzgerald & Jennings
JCO 2015 - Hudson & Ness
PAIN 2009 - Walker
Our paper circa 2017

Note: Applications that do not follow these guidelines will NOT be considered.